Monoclonal antibody, inhibiting the enzymatic activity of vascular endothelial lipase

ABSTRACT

Provided is a monoclonal antibody or a fragment thereof that selectively inhibits the enzymatic activity of vascular endothelial lipase and pharmaceutical compositions containing the same as an active ingredient useful for the treatment of arteriosclerosis or metabolic syndrome.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of copending U.S. application Ser. No.15/611,864, filed on Jun. 2, 2017, which is a Divisional of U.S.application Ser. No. 14/772,340, filed on Sep. 2, 2015 (now U.S. Pat.No. 9,701,757 issued on Jul. 11, 2017) which is the National Phase ofPCT International Application No. PCT/JP2014/056526 filed Mar. 12, 2014,which claims priority on Japanese Patent Application No. 2013-051995filed Mar. 14, 2013, all of which are hereby expressly incorporated byreference into the present application.

TECHNICAL FIELD

The present invention relates to a monoclonal antibody that inhibits theenzymatic activity of vascular endothelial lipase (hereinafter, referredto also EL) and pharmaceutical compositions containing the same. Morespecifically, the present invention relates to an antibody thatselectively inhibits the enzymatic activity of EL, or a part thereof,and a pharmaceutical composition containing the same.

BACKGROUND ART

EL is a phospholipase that belongs to triglyceride lipase (hereinafter,referred to TG) family (non-patent literature: 1). Human EL consists of500 amino acids (NCBI Accession number NP_006024.1, SEQ ID NO: 1) andrabbit EL consists of 500 amino acids (NCBI Accession numberNO_001182567, SEQ ID NO: 2). Lipoprotein lipase (hereinafter, referredto LPL) and hepatic lipase (hereinafter, referred to HL) is contained byTG family.

The analysis of EL knockout mouse and EL transgenic mouse revealed thatEL relates to HDL cholesterol (hereinafter, referred to HDL-c)metabolism by its strong phospholipase activity, and have been a focusas a factor which controls HDL-c level in blood (non-patent literature:2). It has been well-known that there is a negative correlation betweencoronary artery disease (hereinafter, referred to CAD) and HDL-c levelin blood. HDL-c shows anti-artherogenic effect through antioxidanteffect, anti-inflammatory effect and reverse cholesterol transport andso on, low HDL-c emia is recognized as one of the risk factor of CAD.Therefore, EL inhibitor could become a therapeutic agent for CAD throughincreasing HDL-c in blood. In fact, it was reported that lesion mouse ofEL knockout increased HDL-c and decreased atherosclerotic lesions(non-patent literature: 3).

These findings indicate that a selective EL inhibitor is useful astherapeutic agent for abnormality of lipid metabolism andarteriosclerosis.

A selective inhibition of EL is useful as therapeutic agent forabnormality of lipid metabolism and arteriosclerosis, so a production ofEL antibodies which inhibit EL activity is one of the importantapproaches. So far, it has been reported that a rabbit polyclonalantibody which inhibits EL activity was prepared, HDL-c level in mouseblood increased after administrating of the antibody (non-patentliterature: 4).

Polyclonal antibodies recognize various regions of EL and do not have ahigh selectivity against EL. Also, it is impossible to use rabbitanti-EL polyclonal antibodies having high immunogenicity to human astherapeutic agent for chronic diseases because therapeutic agents forchronic diseases such as the abnormality of lipid metabolism andarteriosclerosis related to EL have to be administrated for a long term.Moreover, it is difficult to manipulate immunogenicity of polyclonalantibodies.

By these circumstances, a monoclonal antibody inhibiting selectively theenzymatic activity of EL is awaited.

-   Non-patent document 1: Nature Genetics., 1999, vol. 21, p. 424-   Non-patent document 2: TCM., 2004, vol. 14(5), p. 202-206-   Non-patent document 3: The Journal of Biological Chemistry., 2004,    vol. 279, No. 43, 22 p. 45085-45092-   Non-patent document 4: J. Clin. Invest., 2003, Vol. 111(3), p. 357

SUMMARY OF THE INVENTION Problem to be Solved by the Invention

An objective of the present invention is to provide an antibody thatselectively inhibits enzyme activity of EL, or a an antibody fragmentthereof, and a pharmaceutical composition containing the same.

Means for Solving the Problem

As a result of diligent efforts, the present inventors have succeeded infinding a monoclonal antibody that selectively inhibits the enzymaticactivity of EL.

To be more specific, the present invention relates to:

(1) A monoclonal antibody that inhibits the enzymatic activity ofvascular endothelial lipase or an antibody fragment thereof, wherein itrecognizes at least one amino acid at positions of 1 to 157 in an aminoacid sequence of SEQ ID NO: 1.(2) The monoclonal antibody of (1) or the antibody fragment thereof,wherein it recognizes at least one amino acid at positions of 50 to 100in an amino acid sequence of SEQ ID NO: 1.(3) The monoclonal antibody of (1) or (2) or the antibody fragmentthereof, wherein it recognizes at least one amino acid selected fromarginine at position 50, glutamic acid at position 60, tyrosine atposition 65 and asparagine at position 100 in an amino acid sequence ofSEQ ID NO: 1.(4) The monoclonal antibody of (1) or (2) or the antibody fragmentthereof, wherein it recognizes at least tyrosine at position 65 in anamino acid sequence of SEQ ID NO: 1.(5) The monoclonal antibody of (4) or the antibody fragment thereof,wherein it recognizes at least one amino acid selected from arginine atposition 50, asparagine at position 52, arginine at position 54,aspartic acid at position 58, glutamic acid at position 60, histidine atposition 61, glycine at position 63 and asparagine at position 100 in anamino acid sequence of SEQ ID NO: 1.(6) A monoclonal antibody that inhibits the enzymatic activity ofvascular endothelial lipase or an antibody fragment thereof, wherein itrecognizes at least one amino acid at positions of 202 to 305 in anamino acid sequence of SEQ ID NO: 1.(7) The monoclonal antibody of (6) or the antibody fragment thereof,wherein it recognizes at least one amino acid at positions of 220 to 273in an amino acid sequence of SEQ ID NO: 1.(8) The monoclonal antibody of (6) or (7) or the antibody fragmentthereof, wherein it recognizes at least one amino acid selected fromhistidine at position 220, threonine at position 221, tyrosine atposition 222, threonine at position 223, arginine at position 224,phenylalanine at position 226, glycine at position 227, glycine atposition 231, isoleucine at position 232, glutamine at position 233,methionine at position 234, aspartic acid at position 240, tyrosine atposition 242, proline at position 243, asparagine at position 244,glycine at position 246, glutamine at position 249, proline at position250, glycine at position 251, leucine at position 254, leucine atposition 258, tyrosine at position 263, valine at position 269 andglutamic acid at position 273 in an amino acid sequence of SEQ ID NO: 1.(9) The monoclonal antibody of (6) or (7) or the antibody fragmentthereof, wherein it recognizes at least one amino acid selected fromhistidine at position 220, threonine at position 221, aspartic acid atposition 240, tyrosine at position 242, proline at position 243,asparagine at position 244, glycine at position 246, glutamine atposition 249, proline at position 250, and glycine at position 251 in anamino acid sequence of SEQ ID NO: 1.(10) The monoclonal antibody of (6) or (7) or the antibody fragmentthereof, wherein it recognizes all amino acids of histidine at position220, threonine at position 221, aspartic acid at position 240, tyrosineat position 242, proline at position 243, asparagine at position 244,glycine at position 246, glutamine at position 249, proline at position250 and glycine at position 251 in an amino acid sequence of SEQ ID NO:1.(11) The monoclonal antibody of any one of (1) to (10), wherein itinhibits the enzymatic activity of vascular endothelial lipase with anIC 50 of 10 nM or less.(12) The monoclonal antibody of (11), wherein it inhibits the enzymaticactivity of vascular endothelial lipase with an IC 50 of 5 nM or less.(13) The monoclonal antibody of (11), wherein it inhibits the enzymaticactivity of vascular endothelial lipase with an IC 50 of 2 nM or less.(14) A monoclonal antibody or a fragment thereof, selected from thegroup of

-   1) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 10, amino acid sequence of SEQ        ID NO: 11 and amino acid sequence of SEQ ID NO: 12 and    -   a light chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 14, amino acid sequence of SEQ        ID NO: 15 and amino acid sequence of SEQ ID NO: 16.-   2) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 10, amino acid sequence of SEQ        ID NO: 11 and amino acid sequence of SEQ ID NO: 12 and    -   a light chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said the three CDRs comprising amino acid sequence        of SEQ ID NO: 14, amino acid sequence of SEQ ID NO: 15 and amino        acid sequence of SEQ ID NO: 16, and    -   inhibiting the enzymatic activity of vascular endothelial        lipase.-   3) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said the three CDRs comprising amino acid sequence        of SEQ ID NO: 10, amino acid sequence of SEQ ID NO: 11 and amino        acid sequence of SEQ ID NO: 12, and    -   a light chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 14, amino acid sequence of SEQ        ID NO: 15 and amino acid sequence of SEQ ID NO: 16, and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   4) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said the three CDRs comprising amino acid sequence        of SEQ ID NO: 10, amino acid sequence of SEQ ID NO: 11 and amino        acid sequence of SEQ ID NO: 12, and    -   a light chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said three CDRs comprising amino acid sequence of        SEQ ID NO: 14, amino acid sequence of SEQ ID NO: 15 and amino        acid sequence of SEQ ID NO: 16, and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   5) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 18, amino acid sequence of SEQ        ID NO: 19 and amino acid sequence of SEQ ID NO: 20 and    -   a light chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 22, amino acid sequence of SEQ        ID NO: 23 and amino acid sequence of SEQ ID NO: 24.-   6) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 18, amino acid sequence of SEQ        ID NO: 19 and amino acid sequence of SEQ ID NO: 20 and    -   a light chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said the three CDRs comprising amino acid sequence        of SEQ ID NO: 22, amino acid sequence of SEQ ID NO: 23 and amino        acid sequence of SEQ ID NO: 24, and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   7) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said the three CDRs comprising amino acid sequence        of SEQ ID NO: 18, amino acid sequence of SEQ ID NO: 19 and amino        acid sequence of SEQ ID NO: 20, and    -   a light chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 22, amino acid sequence of SEQ        ID NO: 23 and amino acid sequence of SEQ ID NO: 24, and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   8) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said the three CDRs comprising amino acid sequence        of SEQ ID NO: 18, amino acid sequence of SEQ ID NO: 19 and amino        acid sequence of SEQ ID NO: 20, and    -   a light chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said three CDRs comprising amino acid sequence of        SEQ ID NO: 22, amino acid sequence of SEQ ID NO: 23 and amino        acid sequence of SEQ ID NO: 24, and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   9) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 26, amino acid sequence of SEQ        ID NO: 27 and amino acid sequence of SEQ ID NO: 28 and    -   a light chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 30, amino acid sequence of SEQ        ID NO: 31 and amino acid sequence of SEQ ID NO: 32.-   10) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 26, amino acid sequence of SEQ        ID NO: 27 and amino acid sequence of SEQ ID NO: 28 and    -   a light chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said the three CDRs comprising amino acid sequence        of SEQ ID NO: 30, amino acid sequence of SEQ ID NO: 31 and amino        acid sequence of SEQ ID NO: 32, and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   11) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said the three CDRs comprising amino acid sequence        of SEQ ID NO: 26, amino acid sequence of SEQ ID NO: 27 and amino        acid sequence of SEQ ID NO: 28, and    -   a light chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 30, amino acid sequence of SEQ        ID NO: 31 and amino acid sequence of SEQ ID NO: 32, and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   12) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said the three CDRs comprising amino acid sequence        of SEQ ID NO: 26, amino acid sequence of SEQ ID NO: 27 and amino        acid sequence of SEQ ID NO: 28, and    -   a light chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said three CDRs comprising amino acid sequence of        SEQ ID NO: 30, amino acid sequence of SEQ ID NO: 31 and amino        acid sequence of SEQ ID NO: 32, and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   13) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 34, amino acid sequence of SEQ        ID NO: 35 and amino acid sequence of SEQ ID NO: 36 and    -   a light chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 38, amino acid sequence of SEQ        ID NO: 39 and amino acid sequence of SEQ ID NO: 40.-   14) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 34, amino acid sequence of SEQ        ID NO: 35 and amino acid sequence of SEQ ID NO: 36 and    -   a light chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said the three CDRs comprising amino acid sequence        of SEQ ID NO: 38, amino acid sequence of SEQ ID NO: 39 and amino        acid sequence of SEQ ID NO: 40, and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   15) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said the three CDRs comprising amino acid sequence        of SEQ ID NO: 34, amino acid sequence of SEQ ID NO: 35 and amino        acid sequence of SEQ ID NO: 36, and    -   a light chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 38, amino acid sequence of SEQ        ID NO: 39 and amino acid sequence of SEQ ID NO: 40, and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   16) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said the three CDRs comprising amino acid sequence        of SEQ ID NO: 34, amino acid sequence of SEQ ID NO: 35 and amino        acid sequence of SEQ ID NO: 36, and    -   a light chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said three CDRs comprising amino acid sequence of        SEQ ID NO: 38, amino acid sequence of SEQ ID NO: 39 and amino        acid sequence of SEQ ID NO: 40, and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   17) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 42, amino acid sequence of SEQ        ID NO: 43 and amino acid sequence of SEQ ID NO: 44 and    -   a light chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 46, amino acid sequence of SEQ        ID NO: 47 and amino acid sequence of SEQ ID NO: 48.-   18) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 42, amino acid sequence of SEQ        ID NO: 43 and amino acid sequence of SEQ ID NO: 44 and    -   a light chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said the three CDRs comprising amino acid sequence        of SEQ ID NO: 46, amino acid sequence of SEQ ID NO: 47 and amino        acid sequence of SEQ ID NO: 48, and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   19) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said the three CDRs comprising amino acid sequence        of SEQ ID NO: 42, amino acid sequence of SEQ ID NO: 43 and amino        acid sequence of SEQ ID NO: 44, and    -   a light chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 46, amino acid sequence of SEQ        ID NO: 47 and amino acid sequence of SEQ ID NO: 48, and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   20) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said the three CDRs comprising amino acid sequence        of SEQ ID NO: 42, amino acid sequence of SEQ ID NO: 43 and amino        acid sequence of SEQ ID NO: 44, and    -   a light chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said three CDRs comprising amino acid sequence of        SEQ ID NO: 46, amino acid sequence of SEQ ID NO: 47 and amino        acid sequence of SEQ ID NO: 48, and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   21) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 50, amino acid sequence of SEQ        ID NO: 51 and amino acid sequence of SEQ ID NO: 52 and    -   a light chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 54, amino acid sequence of SEQ        ID NO: 55 and amino acid sequence of SEQ ID NO: 56.-   22) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 50, amino acid sequence of SEQ        ID NO: 51 and amino acid sequence of SEQ ID NO: 52 and    -   a light chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said the three CDRs comprising amino acid sequence        of SEQ ID NO: 54, amino acid sequence of SEQ ID NO: 55 and amino        acid sequence of SEQ ID NO: 56, and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   23) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said the three CDRs comprising amino acid sequence        of SEQ ID NO: 50, amino acid sequence of SEQ ID NO: 51 and amino        acid sequence of SEQ ID NO: 52, and    -   a light chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 54, amino acid sequence of SEQ        ID NO: 55 and amino acid sequence of SEQ ID NO: 56, and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   24) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said the three CDRs comprising amino acid sequence        of SEQ ID NO: 50, amino acid sequence of SEQ ID NO: 51 and amino        acid sequence of SEQ ID NO: 52, and    -   a light chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said three CDRs comprising amino acid sequence of        SEQ ID NO: 54, amino acid sequence of SEQ ID NO: 55 and amino        acid sequence of SEQ ID NO: 56, and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   25) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 58, amino acid sequence of SEQ        ID NO: 59 and amino acid sequence of SEQ ID NO: 60 and    -   a light chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 62, amino acid sequence of SEQ        ID NO: 63 and amino acid sequence of SEQ ID NO: 64.-   26) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 58, amino acid sequence of SEQ        ID NO: 59 and amino acid sequence of SEQ ID NO: 60 and    -   a light chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said the three CDRs comprising amino acid sequence        of SEQ ID NO: 62, amino acid sequence of SEQ ID NO: 63 and amino        acid sequence of SEQ ID NO: 64, and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   27) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said the three CDRs comprising amino acid sequence        of SEQ ID NO: 58, amino acid sequence of SEQ ID NO: 59 and amino        acid sequence of SEQ ID NO: 60, and    -   a light chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 62, amino acid sequence of SEQ        ID NO: 63 and amino acid sequence of SEQ ID NO: 64, and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   28) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said the three CDRs comprising amino acid sequence        of SEQ ID NO: 58, amino acid sequence of SEQ ID NO: 59 and amino        acid sequence of SEQ ID NO: 60, and    -   a light chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said three CDRs comprising amino acid sequence of        SEQ ID NO: 62, amino acid sequence of SEQ ID NO: 63 and amino        acid sequence of SEQ ID NO: 64, and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   29) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 66, amino acid sequence of SEQ        ID NO: 67 and amino acid sequence of SEQ ID NO: 68 and    -   a light chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 70, amino acid sequence of SEQ        ID NO: 71 and amino acid sequence of SEQ ID NO: 72.-   30) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 66, amino acid sequence of SEQ        ID NO: 67 and amino acid sequence of SEQ ID NO: 68 and    -   a light chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said the three CDRs comprising amino acid sequence        of SEQ ID NO: 70, amino acid sequence of SEQ ID NO: 71 and amino        acid sequence of SEQ ID NO: 72, and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   31) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said the three CDRs comprising amino acid sequence        of SEQ ID NO: 66, amino acid sequence of SEQ ID NO: 67 and amino        acid sequence of SEQ ID NO: 68, and    -   a light chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 70, amino acid sequence of SEQ        ID NO: 71 and amino acid sequence of SEQ ID NO: 72, and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   32) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said the three CDRs comprising amino acid sequence        of SEQ ID NO: 66, amino acid sequence of SEQ ID NO: 67 and amino        acid sequence of SEQ ID NO: 68, and    -   a light chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said three CDRs comprising amino acid sequence of        SEQ ID NO: 70, amino acid sequence of SEQ ID NO: 71 and amino        acid sequence of SEQ ID NO: 72, and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   33) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 74, amino acid sequence of SEQ        ID NO: 75 and amino acid sequence of SEQ ID NO: 76 and    -   a light chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 78, amino acid sequence of SEQ        ID NO: 79 and amino acid sequence of SEQ ID NO: 80.-   34) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 74, amino acid sequence of SEQ        ID NO: 75 and amino acid sequence of SEQ ID NO: 76 and    -   a light chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said the three CDRs comprising amino acid sequence        of SEQ ID NO: 78, amino acid sequence of SEQ ID NO: 79 and amino        acid sequence of SEQ ID NO: 80, and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   35) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said the three CDRs comprising amino acid sequence        of SEQ ID NO: 74, amino acid sequence of SEQ ID NO: 75 and amino        acid sequence of SEQ ID NO: 76, and    -   a light chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 78, amino acid sequence of SEQ        ID NO: 79 and amino acid sequence of SEQ ID NO: 80, and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   36) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said the three CDRs comprising amino acid sequence        of SEQ ID NO: 74, amino acid sequence of SEQ ID NO: 75 and amino        acid sequence of SEQ ID NO: 76, and    -   a light chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said three CDRs comprising amino acid sequence of        SEQ ID NO: 78, amino acid sequence of SEQ ID NO: 79 and amino        acid sequence of SEQ ID NO: 80, and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   37) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 82, amino acid sequence of SEQ        ID NO: 83 and amino acid sequence of SEQ ID NO: 84 and    -   a light chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 86, amino acid sequence of SEQ        ID NO: 87 and amino acid sequence of SEQ ID NO: 88.-   38) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 82, amino acid sequence of SEQ        ID NO: 83 and amino acid sequence of SEQ ID NO: 84 and    -   a light chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said the three CDRs comprising amino acid sequence        of SEQ ID NO: 86, amino acid sequence of SEQ ID NO: 87 and amino        acid sequence of SEQ ID NO: 88, and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   39) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said the three CDRs comprising amino acid sequence        of SEQ ID NO: 82, amino acid sequence of SEQ ID NO: 83 and amino        acid sequence of SEQ ID NO: 84, and    -   a light chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 86, amino acid sequence of SEQ        ID NO: 87 and amino acid sequence of SEQ ID NO: 88, and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   40) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said the three CDRs comprising amino acid sequence        of SEQ ID NO: 82, amino acid sequence of SEQ ID NO: 83 and amino        acid sequence of SEQ ID NO: 84, and    -   a light chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said three CDRs comprising amino acid sequence of        SEQ ID NO: 86, amino acid sequence of SEQ ID NO: 87 and amino        acid sequence of SEQ ID NO: 88, and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   41) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 90, amino acid sequence of SEQ        ID NO: 91 and amino acid sequence of SEQ ID NO: 92 and    -   a light chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 94, amino acid sequence of SEQ        ID NO: 95 and amino acid sequence of SEQ ID NO: 96.-   42) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 90, amino acid sequence of SEQ        ID NO: 91 and amino acid sequence of SEQ ID NO: 92 and    -   a light chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said the three CDRs comprising amino acid sequence        of SEQ ID NO: 94, amino acid sequence of SEQ ID NO: 95 and amino        acid sequence of SEQ ID NO: 96, and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   43) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said the three CDRs comprising amino acid sequence        of SEQ ID NO: 90, amino acid sequence of SEQ ID NO: 91 and amino        acid sequence of SEQ ID NO: 92, and    -   a light chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 94, amino acid sequence of SEQ        ID NO: 95 and amino acid sequence of SEQ ID NO: 96, and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   44) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said the three CDRs comprising amino acid sequence        of SEQ ID NO: 90, amino acid sequence of SEQ ID NO: 91 and amino        acid sequence of SEQ ID NO: 92, and    -   a light chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said three CDRs comprising amino acid sequence of        SEQ ID NO: 94, amino acid sequence of SEQ ID NO: 95 and amino        acid sequence of SEQ ID NO: 96, and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   45) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 100, amino acid sequence of        SEQ ID NO: 101 and amino acid sequence of SEQ ID NO: 102 and    -   a light chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 104, amino acid sequence of        SEQ ID NO: 105 and amino acid sequence of SEQ ID NO: 106.-   46) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 100, amino acid sequence of        SEQ ID NO: 101 and amino acid sequence of SEQ ID NO: 102 and    -   a light chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said the three CDRs comprising amino acid sequence        of SEQ ID NO: 104, amino acid sequence of SEQ ID NO: 105 and        amino acid sequence of SEQ ID NO: 106, and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   47) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said the three CDRs comprising amino acid sequence        of SEQ ID NO: 100, amino acid sequence of SEQ ID NO: 101 and        amino acid sequence of SEQ ID NO: 102, and    -   a light chain variable region including three CDRs comprising        amino acid sequence of SEQ ID NO: 104, amino acid sequence of        SEQ ID NO: 105 and amino acid sequence of SEQ ID NO: 106, and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   48) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said the three CDRs comprising amino acid sequence        of SEQ ID NO: 100, amino acid sequence of SEQ ID NO: 101 and        amino acid sequence of SEQ ID NO: 102, and    -   a light chain variable region including three CDRs, said three        CDRs consisting of amino acids that are one or several amino        acids are deleted, substituted or added in at least one of the        three CDRs, said three CDRs comprising amino acid sequence of        SEQ ID NO: 104, amino acid sequence of SEQ ID NO: 105 and amino        acid sequence of SEQ ID NO: 106, and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   (15) A monoclonal antibody or a fragment thereof, selected from the    group of-   1) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acid sequence of        SEQ ID NO: 9, and    -   a light chain variable region comprising amino acid sequence of        SEQ ID NO: 13.-   2) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acid sequence of        SEQ ID NO: 9, and    -   a light chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 13,        and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   3) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 9 and    -   a light chain variable region comprising amino acid sequence of        SEQ ID NO: 13 and inhibiting the enzymatic activity of vascular        endothelial lipase.-   4) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 9 and    -   a light chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 13,        and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   5) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acid sequence of        SEQ ID NO: 17, and    -   a light chain variable region comprising amino acid sequence of        SEQ ID NO: 21.-   6) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acid sequence of        SEQ ID NO: 17, and    -   a light chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 21,        and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   7) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 17        and    -   a light chain variable region comprising amino acid sequence of        SEQ ID NO: 21 and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   8) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 17        and    -   a light chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 21,        and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   9) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acid sequence of        SEQ ID NO: 25, and    -   a light chain variable region comprising amino acid sequence of        SEQ ID NO: 29.-   10) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acid sequence of        SEQ ID NO: 25, and    -   a light chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 29,        and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   11) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 25        and    -   a light chain variable region comprising amino acid sequence of        SEQ ID NO: 29 and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   12) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 25        and    -   a light chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 29,        and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   13) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acid sequence of        SEQ ID NO: 33, and    -   a light chain variable region comprising amino acid sequence of        SEQ ID NO: 37.-   14) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acid sequence of        SEQ ID NO: 33, and    -   a light chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 37,        and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   15) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 33        and    -   a light chain variable region comprising amino acid sequence of        SEQ ID NO: 37 and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   16) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 33        and    -   a light chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 37,        and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   17) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acid sequence of        SEQ ID NO: 41, and    -   a light chain variable region comprising amino acid sequence of        SEQ ID NO: 45.-   18) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acid sequence of        SEQ ID NO: 41, and    -   a light chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 45,        and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   19) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 41        and    -   a light chain variable region comprising amino acid sequence of        SEQ ID NO: 45 and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   20) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 41        and    -   a light chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 45,        and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   21) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acid sequence of        SEQ ID NO: 49, and    -   a light chain variable region comprising amino acid sequence of        SEQ ID NO: 53.-   22) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acid sequence of        SEQ ID NO: 49, and    -   a light chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 53,        and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   23) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 49        and    -   a light chain variable region comprising amino acid sequence of        SEQ ID NO: 5 and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   24) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 49        and    -   a light chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 53,        and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   25) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acid sequence of        SEQ ID NO: 57, and    -   a light chain variable region comprising amino acid sequence of        SEQ ID NO: 61.-   26) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acid sequence of        SEQ ID NO: 57, and    -   a light chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 61,        and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   27) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 57        and    -   a light chain variable region comprising amino acid sequence of        SEQ ID NO: 61 and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   28) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 57        and    -   a light chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 61,        and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   29) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acid sequence of        SEQ ID NO: 65, and    -   a light chain variable region comprising amino acid sequence of        SEQ ID NO: 69.-   30) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acid sequence of        SEQ ID NO: 65, and    -   a light chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 69,        and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   31) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 65        and    -   a light chain variable region comprising amino acid sequence of        SEQ ID NO: 69 and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   32) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 65        and    -   a light chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 69,        and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   33) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acid sequence of        SEQ ID NO: 73, and    -   a light chain variable region comprising amino acid sequence of        SEQ ID NO: 77.-   34) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acid sequence of        SEQ ID NO: 73, and    -   a light chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 77,        and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   35) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 73        and    -   a light chain variable region comprising amino acid sequence of        SEQ ID NO: 77 and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   36) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 73        and    -   a light chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 77,        and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   37) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acid sequence of        SEQ ID NO: 81, and    -   a light chain variable region comprising amino acid sequence of        SEQ ID NO: 85.-   38) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acid sequence of        SEQ ID NO: 81, and    -   a light chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 85,        and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   39) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 81        and    -   a light chain variable region comprising amino acid sequence of        SEQ ID NO: 85 and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   40) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 81        and    -   a light chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 85,        and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   41) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acid sequence of        SEQ ID NO: 89, and    -   a light chain variable region comprising amino acid sequence of        SEQ ID NO: 93.-   42) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acid sequence of        SEQ ID NO: 89, and    -   a light chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 93,        and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   43) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 89        and    -   a light chain variable region comprising amino acid sequence of        SEQ ID NO: 93 and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   44) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 89        and    -   a light chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 93,        and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   45) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acid sequence of        SEQ ID NO: 99, and    -   a light chain variable region comprising amino acid sequence of        SEQ ID NO: 103.-   46) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acid sequence of        SEQ ID NO: 99, and    -   a light chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 103,        and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   47) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 99        and    -   a light chain variable region comprising amino acid sequence of        SEQ ID NO: 103 and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   48) A monoclonal antibody or a fragment thereof,-   having    -   a heavy chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 99        and    -   a light chain variable region comprising amino acids, said amino        acids that are one or several amino acids are deleted,        substituted or added in amino acids sequence of SEQ ID NO: 103,        and-   inhibiting the enzymatic activity of vascular endothelial lipase.-   (16) The pharmaceutical composition for treating or preventing a    disease related to vascular endothelial lipase, comprising the    monoclonal antibody of any one of (1) to (15) or the antibody    fragment thereof.-   (17) The pharmaceutical composition of according to (16), wherein    the disease related to vascular endothelial lipase is Dyslipidemia.-   (18) The monoclonal antibody of any one of (1) to (15) or the    antibody fragment thereof for the treatment or prevention of a    disease related to vascular endothelial lipase.-   (19) The monoclonal antibody of (18) or the antibody fragment    thereof, wherein the disease related to vascular endothelial lipase    is Dyslipidemia.-   (20) A method for the treatment or prevention of a disease related    to vascular endothelial lipase characterized by administering to a    monoclonal antibody of any one of (1) to (15) or the antibody    fragment thereof.-   (21) The method of (20), wherein the disease related to vascular    endothelial lipase is Dyslipidemia.

Effect of the Invention

As a monoclonal antibody of the present invention has the activity forselectively inhibiting enzymatic activity of EL, pharmaceuticalcompositions containing the monoclonal antibody of the present inventionis very useful as a drug, especially a drug for prevention and/ortreatment of dyslipidemia, hyperlipidemia, arteriosclerosis,atherosclerosis, hypercholesterolemia, hypertriglyceridemia, diabetes,obesity and/or syndrome X.

BRIEF EXPLANATION OF DRAWINGS

FIG. 1A shows measurement results of inhibition of enzymatic activity of55A1 antibody against cynomolgus EL, baboon EL and human (1-157) mousechimera EL.

FIG. 1B shows measurement results of inhibiting enzymatic activity of7D4 antibody against cynomolgus EL, baboon EL and human (1-157) mousechimera EL.

FIG. 1C shows measurement results of inhibiting enzymatic activity of14A1 antibody against cynomolgus EL, baboon EL and human (1-157) mousechimera EL.

FIG. 1D shows measurement results of inhibiting enzymatic activity of2D5 antibody against cynomolgus EL, baboon EL and human (1-157) mousechimera EL.

FIG. 1E shows measurement results of inhibiting enzymatic activity of53A11 antibody against cynomolgus EL, baboon EL and human (1-157) mousechimera EL.

FIG. 1F shows measurement results of inhibiting enzymatic activity of13B3 antibody against cynomolgus EL, baboon EL, rabbit EL and human(1-157) mouse chimera EL.

FIG. 1G shows measurement results of inhibiting enzymatic activity of23H8 antibody against cynomolgus EL, baboon EL, rabbit EL and human(1-157) mouse chimera EL.

FIG. 1H shows measurement results of inhibiting enzymatic activity of16B3 antibody against cynomolgus EL, baboon EL, rabbit EL, human (1-157)mouse chimera EL and human (1-305) mouse chimera EL.

FIG. 1I shows measurement results of inhibiting enzymatic activity of16E7 antibody against cynomolgus EL, baboon EL, rabbit EL, human (1-157)mouse chimera EL and human (1-305) mouse chimera EL.

FIG. 1J shows measurement results of inhibiting enzymatic activity of14E1 antibody against cynomolgus EL, baboon EL, rabbit EL, human (1-157)mouse chimera EL and human (1-305) mouse chimera EL.

FIG. 1K shows measurement results of inhibiting enzymatic activity of19E7 antibody against cynomolgus EL, baboon EL, rabbit EL, human (1-157)mouse chimera EL and human (1-305) mouse chimera EL.

FIG. 2A shows measurement results of inhibiting enzymatic activity of55A1 antibody against human HL and human LPL.

FIG. 2B shows measurement results of inhibiting enzymatic activity of7D4 antibody against human HL and human LPL.

FIG. 2C shows measurement results of inhibiting enzymatic activity of14A1 antibody against human HL and human LPL.

FIG. 2D shows measurement results of inhibiting enzymatic activity of2D5 antibody against human HL and human LPL.

FIG. 2E shows measurement results of inhibiting enzymatic activity of53A11 antibody against human HL and human LPL.

FIG. 2F shows measurement results of inhibiting enzymatic activity of13B3 antibody against human HL and human LPL.

FIG. 2G shows measurement results of inhibiting enzymatic activity of23H8 antibody against human HL and human LPL.

FIG. 2H shows measurement results of inhibiting enzymatic activity of16B3 antibody against human HL and human LPL.

FIG. 2I shows measurement results of inhibiting enzymatic activity of16E7 antibody against human HL and human LPL.

FIG. 2J shows measurement results of inhibiting enzymatic activity of14E1 antibody against human HL and human LPL.

FIG. 2K shows measurement results of inhibiting enzymatic activity of19E7 antibody human HL and human LPL.

FIG. 2L shows measurement results of inhibiting enzymatic activity of16F6 antibody human HL and human LPL.

FIG. 3A shows measurement results of binding activity of 55A1 antibodyagainst cynomolgus EL, baboon EL, rabbit EL, mouse EL and human (1-157)mouse chimera EL.

FIG. 3B shows measurement results of inhibiting enzymatic activity of7D4 antibody against cynomolgus EL, baboon EL, rabbit EL, mouse EL andhuman (1-157) mouse chimera EL.

FIG. 3C shows measurement results of inhibiting enzymatic activity of14A1 antibody against cynomolgus EL, baboon EL, rabbit EL, mouse EL andhuman (1-157) mouse chimera EL.

FIG. 3D shows measurement results of inhibiting enzymatic activity of2D5 antibody against cynomolgus EL, baboon EL, rabbit EL, mouse EL andhuman (1-157) mouse chimera EL.

FIG. 3E shows measurement results of inhibiting enzymatic activity of 53A11 antibody against cynomolgus EL, baboon EL, rabbit EL, mouse EL andhuman (1-157) mouse chimera EL.

FIG. 3F shows measurement results of inhibiting enzymatic activity of13B3 antibody against cynomolgus EL, baboon EL, rabbit EL, mouse EL andhuman (1-157) mouse chimera EL.

FIG. 3G shows measurement results of inhibiting enzymatic activity of23H8 antibody against cynomolgus EL, baboon EL, rabbit EL, mouse EL andhuman (1-157) mouse chimera EL.

FIG. 3H shows measurement results of inhibiting enzymatic activity of16B3 antibody against cynomolgus EL, baboon EL, rabbit EL, mouse EL,human (1-157) mouse chimera EL, human (1-305) mouse chimera EL and human(202-500) mouse chimera EL.

FIG. 3I shows measurement results of inhibiting enzymatic activity of16E7 antibody against cynomolgus EL, baboon EL, rabbit EL, mouse EL,human (1-157) mouse chimera EL, human (1-305) mouse chimera EL and human(202-500) mouse chimera EL.

FIG. 3J shows measurement results of inhibiting enzymatic activity of14E1 antibody against cynomolgus EL, baboon EL, rabbit EL, mouse EL,human (1-157) mouse chimera EL, human (1-305) mouse chimera EL and human(202-500) mouse chimera EL.

FIG. 3K shows measurement results of inhibiting enzymatic activity of19E7 antibody against cynomolgus EL, baboon EL, rabbit EL, human (1-157)mouse chimera EL and human (1-305) mouse chimera EL.

FIG. 3L shows measurement results of inhibiting enzymatic activity of16F6 antibody against cynomolgus EL, human (1-305) mouse chimera EL andhuman (187-500) mouse chimera EL.

FIG. 4A (SEQ ID NOS: 9 and 13) shows amino acid sequence of a variableregion of 55A1 antibody.

FIG. 4B (SEQ ID NOS: 17 and 21) shows amino acid sequence of a variableregion of 7D4 antibody.

FIG. 4C (SEQ ID NOS: 25 and 29) shows amino acid sequence of a variableregion of 14A1 antibody.

FIG. 4D (SEQ ID NOS: 33 and 37) shows amino acid sequence of a variableregion of 2D5 antibody.

FIG. 4E (SEQ ID NOS: 41 and 45) shows amino acid sequence of a variableregion of 53A11 antibody.

FIG. 4F (SEQ ID NOS: 49 and 53) shows amino acid sequence of a variableregion of 13B3 antibody.

FIG. 4G (SEQ ID NOS: 57 and 61) shows amino acid sequence of a variableregion of 23H8 antibody.

FIG. 4H (SEQ ID NOS: 65 and 69) shows amino acid sequence of a variableregion of 16B3 antibody.

FIG. 4I (SEQ ID NOS: 73 and 77) shows amino acid sequence of a variableregion of 16E7 antibody.

FIG. 4J (SEQ ID NOS: 81 and 85) shows amino acid sequence of a variableregion of 14E1 antibody.

FIG. 4K (SEQ ID NOS: 89 and 93) shows amino acid sequence of a variableregion of 19E7 antibody.

FIG. 4L (SEQ ID NOS: 99 and 103) shows amino acid sequence of a variableregion of 16F6 antibody.

FIG. 5A shows an alignment of the amino acid sequences of heavy chainvariable region of 55A1 antibody (SEQ ID NO: 9), 7D4 antibody (SEQ IDNO: 17), 14A1 antibody (SEQ ID NO: 25) and 2D5 antibody (SEQ ID NO: 33).

FIG. 5B shows an alignment of the amino acid sequences of light chainvariable region of 55A1 antibody (SEQ ID NO: 13), 7D4 antibody (SEQ IDNO: 21), 14A1 antibody (SEQ ID NO: 29) and 2D5 antibody (SEQ ID NO: 37).

FIG. 5C shows an alignment of the amino acid sequences of heavy chainvariable region of 53A11 antibody (SEQ ID NO: 41), 13B3 antibody (SEQ IDNO: 49) and 23H8 antibody (SEQ ID NO: 57).

FIG. 5D shows an alignment of the amino acid sequences of light chainvariable region of 53A11 antibody (SEQ ID NO: 45), 13B3 antibody (SEQ IDNO: 53) and 23H8 antibody (SEQ ID NO: 61).

FIG. 6A shows an alignment of the amino acid sequences of heavy chainvariable region of 16B3 antibody (SEQ ID NO: 65) and 16E7 antibody (SEQID NO:73).

FIG. 6B shows an alignment of the amino acid sequences of light chainvariable region of 16B3 (SEQ ID NO: 60) antibody and 16E7 antibody (SEQID NO: 77).

FIG. 6C shows an alignment of the amino acid sequences of heavy chainvariable region of 14E1 antibody (SEQ ID NO: 81) and 19E7 antibody (SEQID NO: 89).

FIG. 6D shows an alignment of the amino acid sequences of light chainvariable region of 14E1 antibody (SEQ ID NO: 85) and 19E7 antibody (SEQID NO: 93).

FIG. 7A shows binding activity of 55A1 antibody against baboon EL thatwas introduced mutation.

FIG. 7B shows binding activity of 7D4 antibody against baboon EL thatwas introduced mutation.

FIG. 7C shows binding activity of 14A1 antibody against baboon EL thatwas introduced mutation.

FIG. 7D shows binding activity of 2D5 antibody against baboon EL thatwas introduced mutation.

FIG. 7E shows binding activity of 53A11 antibody against baboon EL thatwas introduced mutation.

FIG. 7F shows binding activity of 13B3 antibody against baboon EL thatwas introduced mutation.

FIG. 7G shows binding activity of 23H8 antibody against baboon EL thatwas introduced mutation.

FIG. 7H shows binding activity of 16B3 antibody against baboon EL thatwas introduced mutation.

FIG. 7I shows binding activity of 16E7 antibody against baboon EL thatwas introduced mutation.

FIG. 7J shows binding activity of 14E1 antibody against baboon EL thatwas introduced mutation.

FIG. 7K shows binding activity of 19E7 antibody against baboon EL thatwas introduced mutation.

MODE FOR CARRYING OUT THE INVENTION

The present invention provides a monoclonal antibody characterized byselectively inhibiting the enzymatic activity of vascular endotheliallipase. As the monoclonal antibody of the present invention has theactivity for selectively inhibiting enzymatic activity of vascularendothelial lipase, it is very useful as a drug for prevention ortreatment of arteriosclerosis or metabolic syndrome.

It is important to use peptide having consecutive amino acid residuescontaining amino acid sequence at positions of 1 to 157 or 202 to 305region in amino acid sequence of SEQ ID NO: 1 as antigen to produce amonoclonal antibody of the present invention. The length is notparticularly limited, but six or more residues which have immunogenicityare desired. We can use naturally or artificially highly expressed celllines, these membrane fractions, these purified products, fusionproteins with other proteins or peptides (for examples, tag proteinssuch as FLAG-tag, HIS-tag, GST-tag or C2tag etc. or fluorescent proteinssuch as GFP or EGFP etc.), or chemically synthesized peptides asspecific examples of these antibodies. In addition, preparation methodsof these immunogens are known to those skilled in the art.

The monoclonal antibody of the present invention may be prepared by anknown commonly used production method. Concretely, a mammal, preferably,mouse, rat, hamster, guinea pig, rabbit, cat, dog, pig, goat, sheep,donkey, horse or bovine, more preferably mouse, rat, hamster, guinea pigor rabbit is immunized with an immunogen of the present invention,together with Freund's adjuvant as necessary, by one or several times ofsubcutaneous, intramuscular, intravenous, intrafootpad orintraperitoneal injection. Usually, immunization is conducted once tofour times every about 1 to 21 days after primary immunization, andantibody producing cells may be acquired from the immunized mammal afterabout 1 to 10 days from the final immunization. The number of times andtime interval of immunization may be appropriately changed depending onthe property of the immunogen being used.

Hybridoma that secrets monoclonal antibody may be prepared according tothe Kohler and Milstein's method (Nature, 1975, vol. 256, p. 495-497)and a corresponding method. That is, hybridoma may be prepared by cellfusion between an antibody producing cell contained in spleen, lymphnode, bone marrow, tonsil or the like, preferably in spleen acquiredfrom a mammal immunized as described above, and a myeloma cell lackingautoantibody producing ability derived, preferably from a mammal such asmouse, rat, guinea pig, hamster, rabbit or human, more preferably frommouse, rat or human.

As a myeloma cell used in cell fusion, generally, cell lines obtainedfrom mouse, for example, P3-U1, NS-1, SP-2, 653, X63, AP-1 and the likemay be used.

Hybridoma that produces monoclonal antibody is screened by culturing ahybridoma, for example, in a microtiter plate, measuring reactivity toan immunogen used in mouse immunization as described above in culturesupernatant in the well where proliferation is observed, by a measuringmethod such as RIA, ELISA or FACS and selecting a clone that produces amonoclonal antibody exhibiting specific affinity with the immunogen orhapten. When measuring the said reactivity, an immunogen is usuallysolid-phased, and an antibody in culture supernatant that binds to thesolid-phased immunogen is detected by an anti-mouse secondary antibodylabeled with a radioactive substance, a fluorescent substance or anenzyme. Further in the case of using the cells expressing the immunogen,we add the hybridoma culture supernatant to the cells, then afterreacting with secondary antibodies labeled with a fluorescent, we candetect a monoclonal antibody of the present invention that binds to theimmunogen on the cell membrane by measuring fluorescence intensity ofthe cells with fluorescence detection apparatus such as flow cytometryor the like.

Production of monoclonal antibody from selected hybridoma may beachieved by culturing hybridoma in vitro or in ascites of mouse, rat,guinea pig, hamster or rabbit, preferably of mouse or rat, or morepreferably of mouse, followed by isolation from the obtained culturesupernatant or ascites of mammal. In the case of in vitro culture, thehybridoma may be cultured in a known nutrient medium or in any nutrientcultures derived and prepared from a known base medium used forproliferating, maintaining and storing hybridoma and for producingmonoclonal antibody in culture supernatant, depending on variousconditions such as property of cultured cell species, object of the testresearch and culturing method.

As a base medium, for example, low-calcium media such as Ham′F12 medium,MCDB153 medium or low-calcium MEM culture, and high-calcium media suchas MCDB104 medium, MEM medium, D-MEM medium, RPMI1640 medium, AF104medium, or RD medium can be recited, and such a base medium may contain,for example, serum, hormone, cytokine and/or various inorganic ororganic substances depending on the object.

Isolation and purification of monoclonal antibody may be achieved bysubjecting the culture supernatant or ascites as described above tosaturated ammonium sulfate, ion exchange chromatography (e.g., DEAE orDE52), affinity column chromatography such as anti-immunoglobulin columnor protein A column or the like.

As a monoclonal antibody of the present invention, a recombinantantibody that is produced using gene recombination technique in such amanner that an antibody gene is cloned from antibody producing cell, forexample, hybridoma, and incorporated into an appropriate vector, and thevector is introduced into a host may be used (for example, Carl et al.,THERAPEUTIC MONOCLONAL ANTIBODIES, published in 1990).

Concretely, from a hybridoma that produces an objective antibody, orfrom an immune cell that produces an antibody, for example, from a cellobtained by immortalizing sensitized lymphocyte or the like by cancergene or the like, mRNA encoding a variable region (V region) of antibodyis isolated. In isolation of mRNA, whole RNA is prepared by a knownmethod, for example, by guanidine ultracentrifugation (Chirgwin, J. M.et al., Biochemistry (1979) 18, 5294-5299) or the like, and mRNA isprepared by using mRNA Purification Kit (available from Pharmacia) orthe like.

From the obtained mRNA, cDNA of antibody V region is synthesized using areverse transcriptase. Synthesis of cDNA may be conducted using AMVReverse Transcriptase First-strand cDNA Synthesis Kit or the like.Further, for synthesis and amplification of cDNA, 5′-Ampli FINDERRACEKit (available from Clonetech) and 5′-RACE method using PCR(Frohman, M. A. et al, Proc. Natl. Acad. Sci. USA 1988, vol. 85, p.8998) may be used. An objective DNA fragment is purified from theobtained PCR product, and connected with vector DNA. A recombinantvector is thus created and introduced into E. coli or the like, and acolony is selected and a desired recombinant vector is prepared. DNAbase sequence of objective DNA is verified by a known method, forexample, by deoxy method.

If DNA encoding V region of objective antibody is obtained, the DNA isconnected with DNA encoding a desired antibody constant region (Cregion), and incorporated into an expression vector. Alternatively, DNAencoding V region of antibody may be incorporated into an expressionvector containing DNA of antibody C region. For production of antibodyused in the present invention, antibody gene is incorporated into anexpression vector in such a manner that it is expressed under control ofan expression control region, for example, enhancer/promoter. Next, ahost cell can be transformed with this expression vector to causeexpression of antibody.

For expression of antibody gene, heavy chain (H chain) or light chain (Lchain) of antibody may be separately incorporated into expressionvectors, or a host may be co-transformed with these expression vectors,or DNA encoding H chain and L chain may be incorporated into a singleexpression vector to transform a host with the resultant expressionvector (see WO94/11523).

Preparation method of a monoclonal antibody of the present inventionother than the above can be also used so called phage displaytechnology. Concretely, for example antibody gene library prepared as amaterial human or animal (for example, rabbit, mouse, rat, hamster orthe like) B lymphosate by known method or completely synthesizedantibody gene library prepared from selected and modified human oranimal germ line sequence is presented to the cell surface, on theribosome or the like of bacteriophage, Escherichia coli, yeast, animalcells or the like. In this case, the forms of the antibody to bepresented on the cell surface are listed IgG molecules, IgM molecules,Fab fragments, single chain Fv (scFv) fragments, etc.

We can obtain antibody genes by rearranging thus obtained monoclonalantibody fragment to the corresponding region of the IgG antibody geneby a known method. And we incorporate thus genes obtained in this mannerinto a suitable vector, introduce the vector into the host, we canprepare the antibody with recombinant DNA techniques (for examples, seeCarl et. al. THERAPEUTIC MONOCLONAL ANTIBODIES, 1990 issue).

The monoclonal antibody of the present invention is characterized inbeing selective for the inhibition of the enzymatic activity of vascularendothelial lipase. Below, we show an example of a procedure formeasuring ability of inhibiting the enzymatic activity of EL.

The DNA encoding EL is cloned into pcDNA3.1 expression vector(Invitrogen). The expression vector is transfected into HEK293F cellsand culture at 37° C., 8% CO2 for 2 days. The cell cultures arecentrifuged, the cells are collected, the cells are suspended with PBScontaining 20 U/mL of Heparin. The cell suspension is incubated at 37°C. for 45 min. The supernatant obtained by removing cells withcentrifugation is used as human EL enzyme solution to measure inhibitoryactivity.

After adding a monoclonal antibody to the solution containing 20 mMTris-HCl Buffer (pH 7.5), 0.5% bovine serum albumin, 4 mM CaCl2, 150 mMNaCl and 2 mg/mL human HDL (Athens Research&Techonology), EL enzymesolution is added. After reaction at 37° C. for 2 hr, free fatty acid(NEFA) made from HDL by EL enzyme is determined using NEFA C-test Wako(Wakojyunyakukougyo), the NFFA amount is used as enzyme activity index.Enzyme activity in the case of adding no antibody was determined ascontrol value and the specific activity is calculated against thecontrol value at each concentration of the antibody. The concentrationwhere 50% of the antibody is inhibited can be calculated from theinhibition curve.

Effective concentration (IC50) of antibody which shows 50% inhibition ofEL enzyme activity is often used as an indicator of EL inhibitoryactivity.

In the present invention, a monoclonal antibody or a fragment thereofinhibiting the enzymatic activity of vascular endothelial lipase with anIC 50 of 10 nM or less means a monoclonal antibody or a fragment thereofshowing IC 50 of 10 nM or less measured by method of said [0027]-[0029].Any species EL may be used for the measurement if the EL is derived froma mammal. If a monoclonal antibody or a fragment thereof inhibits theenzymatic activity of EL of any kind of mammal species with an IC 50 of10 nM or less, the monoclonal antibody or a fragment thereof is amonoclonal antibody or a fragment thereof showing IC 50 of 10 nM orless.

And the monoclonal antibody of the present invention is characterized inbeing selective for the inhibition of the enzymatic activity of EL.Below, we show an example of a confirmation procedure for measuringability of selectively inhibiting the enzymatic activity of EL, in otherwords, not inhibiting the enzymatic activity of LPL or HL.

The DNA encoding HL is cloned into pcDNA3.1 expression vector(Invitrogen). The expression vector is transfected into HEK293F cellsand culture at 37° C., 8% CO2 for 2 days. The cell cultures arecentrifuged, the cells are collected, the cells are suspended with PBScontaining 20 U/mL of Heparin. The cell suspension is incubated at 37°C. for 45 min. The supernatant obtained by removing cells withcentrifugation is used as human HL enzyme solution. LPL enzyme solutionis prepared by using the same procedures. After adding a monoclonalantibody to the solution containing 20 mM Tris-HCl Buffer (pH 7.5), 0.5%bovine serum albumin, 4 mM CaCl2, 150 mM NaCl and 0.5 mg/mL human VLDL(INTRACEL), HL or LPL enzyme solution is added. After reaction at 37° C.for 2 hr, free fatty acid (NEFA) made from VLDL by HL or LPL enzyme isdetermined using NEFA C-test Wako (Wakojyunyakukougyo), the NFFA amountis used as enzyme activity index. Enzyme activity in the case of addingno antibody was determined as control value and the specific activity iscalculated against the control value at each concentration of antibody.

Being selective for the inhibition of the enzymatic activity of EL meansinhibiting not more than 3% LPL or HL enzymatic activities, when themonoclonal antibody are added at a concentration equal to IC50 againstEL. An epitope region of a monoclonal antibody of the present inventionis preferably the region of EL that has not homology with LPL or HLbecause a monoclonal antibody of the present invention is characterizedby not inhibiting enzyme activity of LPL and HL, and is characterized inbeing selective for the inhibition of enzyme activity of EL.

The monoclonal antibody of the present invention includes recombinantmonoclonal antibodies that are artificially modified for the purpose oflowering heterologous antigenicity against human, for example, chimeramonoclonal antibody, humanized monoclonal antibody and human monoclonalantibody.

The monoclonal antibody of the present invention may be a conjugateantibody bound to various molecules such as polyethylene glycol (PEG),radioactive material, toxin. These conjugate antibodies can be obtainedby chemically modifying obtained antibodies. The techniques ofmodification of antibody are well known in this field. These conjugateantibodies are included in monoclonal antibodies of the presentinvention.

And the monoclonal antibody of the present invention may fuse to theother proteins at the N terminal or C terminal of the antibody (ClinicalCancer Research, 2004, 10, 1274-1281). Those skilled in the art mayproperly select fusion protein.

In the present invention, “a monoclonal antibody fragment” means a partof the above-mentioned monoclonal antibody of the present invention andhas the specific binding ability to vascular endothelial lipase as withthe monoclonal antibody, or means a fragment that has the specificbinding ability to vascular endothelial lipase as with the monoclonalantibody and has the effect of the inhibiting enzymatic activity ofvascular endothelial lipase as with the monoclonal antibody. Concretely,fragments that have specific associativity against EL are listed Fab,F(ab′)2, Fab′, single chain antibody (scFv), disulfide stabilizedantibody (dsFv), dimerized V region fragment (Diabody), peptidecontaining CDR, etc. (Expert opinion on therapeutic patents, vol. 6, No.5, p. 441-456, 1996).

A monoclonal antibody of the present invention or a fragment thereof isuseful as a pharmaceutical composition. Therefore, a pharmaceuticalcomposition containing a monoclonal antibody of the present invention ora fragment thereof may be administered systemically or topically by oralor parenteral route. For parenteral administration, for example,intravenous injection such as drip infusion, intramuscular injection,intraperitoneal injection, subcutaneous injection, intranasaladministration, inhalation and the like can be selected.

Also, a monoclonal antibody of the present invention is applicable tothe diagnostic for dyslipidemia, hyperlipidemia, arteriosclerosis,atherosclerosis, hypercholesterolemia, hypertriglyceridemia, diabetes,obesity and/or syndrome X because a monoclonal antibody of the inventionhas the specific binding ability to against vascular endothelial lipase.

A monoclonal antibody of the present invention or a fragment thereof ischaracterized of recognizing at least one amino acid at positions of 1to 157 in an amino acid sequence of SEQ ID NO: 1 or recognizing at leastone amino acid at positions of 202 to 305 in an amino acid sequence ofSEQ ID NO: 1. Recognizing at least one amino acid at positions of 1 to157 in an amino acid sequence of SEQ ID NO: 1 means that a monoclonalantibody or a fragment thereof recognizes at least one amino acid inthis region.

An patient of the pharmaceutical composition of the present invention isassumed arteriosclerosis and metabolic syndrome. Effective dose isselected in the range of 0.01 mg to 100 mg per 1 kg of body weight perone time. Alternatively, a dose of 5 to 5000 mg, preferably a dose of 10to 500 mg per a patient may be selected. However, a dose of thepharmaceutical composition containing the monoclonal antibody of thepresent invention or a fragment thereof is not limited to these doses.Administering duration may be also appropriately selected depending onthe age, symptom and the like of the patient. The pharmaceuticalcomposition of the present invention may also include a pharmaceuticallyacceptable carrier or additive as well depending on the route ofadministration. Examples of such carrier and additive include water,pharmaceutically acceptable organic solvent, collagen, polyvinylalcohol, polyvinylpyrrolidone, sodium alginate, water-soluble dextran,pectin, methyl cellulose, ethyl cellulose, casein, diglycerin, propyleneglycol, polyethylene glycol, Vaseline, human serum albumin (HSA),mannitol, sorbitol, lactose, and surfactants permitted as apharmaceutical additive. An additive for use is appropriately selectedor combined from the above depending on the dose form, but, it is notlimited thereto.

The present invention is described below in more detail by the way ofexamples. However, the present invention is not limited to the followingexamples. Unless specifying otherwise as a procedure for preparingantibody, we used methods described in Immunochemistry in Practice(Blackwell Scientific Publications). Also unless specifying otherwise asthe genetic engineering techniques, we used methods described inMolecular Cloning: A Laboratory Manual, 2^(nd) Edition (Cold SpringHarbor Laboratory).

Example 1

Preparation of Recombinant Adenovirus to Express Baboon EL

The cDNA of baboon EL with C2 tag was cloned into pShuttle vector(Clontech). This sub-cloned vector and the vector carrying adenoviralbackbone gene was digested by PI-SceI and I-CeuI enzyme (Adeno-xAccessory Kit, Clontech). The ligation reaction of the digestedfragments was conducted at 16° C. for 3 hrs (Ligation high, TOYOBO) andthe ligation products were transformed to E. coli (OneShot stbl3Chemically Competent, Invitrogen). After selection of Ampicillin,plasmid DNA was purified from obtained clone (QIAprep spin Miniprep Kit,QIAGEN) and was digested by PacI enzyme to cut E. coli growth area (NewEngland Biolabs). With the above, plasmid DNA was acquired to generateadenovirus vector. Acquired plasmid DNA was transfected to HEK293 cells(American Type Culture Collection) using Lipofectamine 2000 (Invitrogen)and cultured in DMEM containing 10% FBS at 37° C. After transfection, wechanged culture medium every 5 days and we continued to culture cellsuntil confirming cytopathic effect (CPE). After confirming CPE, thecells and culture supernatant were collected. After the cells weresubjected to five rounds of freeze/thaw with dry ice-methanol bath andwarm bath, supernatant which was obtained by 15 min centrifugation wascollected as cell extracts. The culture supernatant was mixed with theculture medium and used as a primary virus stock. Amplification of thevirus stock was achieved by adding the virus stock to HEK293 cells andrepeating same procedures. After amplification of the virus stock, thefinally obtained cell extracts was treated with Benzonase(Merck-Novagen) for 30 min at 37° C., then supernatant was used for thepurification of viral vector by following density gradientcentrifugation. We overlaid PBS containing 1.5, 1.35, 1.25 g/cm³ cesiumchloride into the centrifuging tube, then overlaid the supernatant. Wecentrifuged this at 35,000 rpm for 1 hr at 16° C., and collectedobtained virus vector by visual. Collected viral vector was dialyzedagainst PBS containing 10% glycerol, and then used as purifiedadenoviral vectors. A part of viral vector was used for titration(Adeno-X rapid titer kit, Clontech) and self-proliferative potentialgain-of-emergence decision, and only used to immunize the following onlythose without abnormal. The amino acid sequence of baboon EL-C2 tag wasdescribed in SEQ ID NO: 2.

Example 2

Preparation of EL Heparin Extract

The DNA encoding baboon EL was cloned into pcDNA3.1 expression vector(Invitrogen). The expression vector was transfected into HEK293F cellsand cultured at 37° C., 8% CO2 for 2 days. The cell culture wascentrifuged and the cells were collected, the cells were suspended withPBS containing 20 U/mL of Heparin. The cell suspension was incubated at37° C. for 45 min. The supernatant obtained by removing cells withcentrifugation was used as baboon EL enzyme solution. Using the samemethod, cynomolgus monkey EL, human [1-157]-mouse chimeric EL, human[1-53]-mouse chimeric EL, human [61-111]-mouse chimeric EL, rabbit EL,mouse EL, human [1-305]-mouse chimeric EL and human [202-500]-mousechimeric EL heparin extract were prepared. The amino acid sequences ofcynomolgus monkey EL, human [1-157-mouse chimeric EL, human [1-53]-mousechimeric EL, human [61-111]-mouse chimeric EL, rabbit EL, mouse EL,human [1-305]-mouse chimeric EL and human [202-500]-mouse chimeric ELwith C2 tag were described SEQ ID NO: 2, 3, 4, 5, 6, 7, 96 and 97.

Example 3

Immunization of Baboon EL Expression Adenoviral Vector

6 week-old female mice (A/J Jms Slc spices, obtained from Nihon SLC)were immunized intravenously, subcutaneously or intramuscularly with2×10⁹ i.f.u. adenovirus vector carrying baboon EL gene. Every 7 daysafter administration, the blood sample was taken from tail vein and theantibody titer was measured. And, additional administration ofadenovirus vector carrying baboon EL gene was done intravenously,subcutaneously or intramuscularly. The mice which showed high titer werebooster immunized from tail vein as the final administration.

Example 4

Production of Hybridoma Producing Antibodies

The abdominal cavity of mouse which showed high titer was opened and thespleen was isolated three days after final immunization. Spleen cellsand mouse myeloma cells (p3x63-Ag8.U1, Tokyo tumor laboratories) werefused using 50% polyethylene glycol 4000, and hybridoma cells wereselected in a culture medium containing hypoxanthine, aminopterin andthymidine.

Example 5

Screening of a Hybridoma Cells which Produces Anti-Baboon EL Antibodies

Ten days after the cell fusion, hybridoma cells which producedanti-baboon EL antibodies were selected. Each well of 384 wellmicrotiter plates (Nunc) was immobilized with 35 μL of Tris/HCl buffer(50 mM Tris/HCl, pH 7.5) containing 0.35 μg of anti-mouse IgG-Fc(Jackson Immuno Research). The plates were incubated at 4° C. for 16 hr.After washing the wells one time with 90 μL of washing solution (salinecontaining 0.01% Tween20), 100 μL of Block-Ace (Dainihonsumitomo) wasadded to the wells and incubated at room temperature for 2 hr(immobilized plate of anti-mouse IgG-Fc antibody). After washing thewells three times with 90 μL of washing buffer, 15 μL of assay buffercontaining baboon EL heparin extract (50 mM Tris/HCl, PH 7.4 containing4% Block-Ace, 0.05% Tween20, 150 mM NaCl) were added to the wells andincubated at room temperature at 4° C. for 16 hr. After washing thewells three times with 90 μL of washing buffer, 15 μL assay buffercontaining biotin-labeled anti-C2-tag antibody and HRP-labeledStreptavidin (Thermo scientific) were added to the wells and incubatedat room temperature for 1 hr. After washing the wells three times with90 μL of washing buffer, 15 μL of TMB+-Substrate—Chromogen (DAKO) wasadded and incubated at room temperature for 30 min. The reaction wasstopped with adding 15 μL of 0.05 M H2SO₄ and then measured absorbance450 nm. From the result of screening, the 11 hybridomas (55A1, 7D4,14A1, 2D5, 53 A11, 13B3, 23H8, 16B3, 16E7, 14E1 and 19E7) which producedanti-baboon EL antibody was selected. The antibody which was produced byhybridoma of 55A1, 7D4, 14A1, 2D5, 53 A11, 13B3, 23H8, 16B3, 16E7, 14E1and 19E7 were named respectively 55A1, 7D4, 14A1, 2D5, 53A11, 13B3,23H8, 16B3, 16E7, 14E1 and 19E7 antibody. The IgG subclasses weredetermined using Mouse Immunoglobulin Isotyping Kit (BD Biosciences).

The subclass of 55A1, 2D5, 53A11, 13B3, 23H8, 16B3 and 19E7 was IgG2a.The subclass of 7D4, 14A1, 16E7 and 14E1 was IgG1.

Example 6

Measurement of Inhibitory Activity of Anti-EL Antibodies Against EL

The inhibitory activity of anti-EL antibodies (55A1, 7D4, 14A1, 2D5,53A11, 13B3, 23H8, 16B3, 16E7, 14E1 and 19E7) against baboon EL,cynomolgus monkey EL, human (1-157) mouse chimeric EL, human (1-53)mouse chimeric EL, human (61-111) mouse chimeric EL, rabbit EL, mouseEL, human (1-305) mouse chimeric EL and human (202-500) mouse chimericEL were measured as follows. Anti-EL antibody solution, assay buffer (20mM Tris/HCl (pH 7.5), 0.5% BSA, 4 mM CaCl₂, 150 mM NaCl) and 2 mg/mLhuman HDL (Athens Research & Technology) were mixed in a microtiterplate, followed by adding EL heparin extract. After incubation at 37° C.for 90 min, non-esterified Fatty Acid (NEFA) released from HDL wasdetermined using a commercially available kit (NEFA C test-Wako, Wako).The NEFA amount was used as enzyme activity index. Enzyme activity inthe case of adding no anti-EL antibody was determined as control valueand specific activity was calculated against the control value at eachconcentration of the anti-EL antibody (FIG. 1A-K). The concentration ofanti-EL antibody where 50% of EL activity was inhibited was calculatedfrom the inhibition curve as IC50 value.

Example 7

Measurement of Inhibitory Activity of Anti-EL Antibody Against HL andLPL

The DNA encoding human HL was cloned into pcDNA3.1 expression vector(Invitrogen). The expression vector was transfected into HEK293F cellsand cultured at 37° C., 8% CO2 for 2 days. The cells were centrifugedand the cells ware collected, the cells were suspended with PBScontaining 20 U/mL of Heparin (SIGMA). The cell suspension was incubatedat 37° C. for 45 min. The supernatant obtained by removing cells withcentrifugation was used as human HL enzyme solution. Human LPL enzymesolution was prepared by using the same method. After adding anti-ELantibody to the solution containing 20 mM Tris-HCl Buffer (pH 7.5), 0.5%bovine serum albumin, 4 mM CaCl₂, 150 mM NaCl and 0.5 mg/mL human VLDL(INTRACEL), human HL or human LPL enzyme was added (total volume 10 μl).After reaction at 37° C. for 2 hr, free fatty acid (NEFA) made from VLDLby HL or LPL enzyme was determined using NEFA C-test Wako (Wako), theNFFA amount was used as enzyme activity index. Enzyme activity in thecase of adding no anti-EL antibody was determined as control value andthe specific activity was calculated against the control value at eachconcentration of the antibody (FIG. 2A-K). For comparison, the resultwas described side by side inhibition curve of cynomolgus monkey EL. Asa result, it was shown that 55A1, 7D4, 14A1, 2D5, 53A11, 13B3, 23H8,16B3, 16E7, 14E1, 19E7 and 16F6 antibodies didn't inhibit neither HL norLPL enzyme activity.

Example 8

Measurement of Binding Activity of Anti-EL Antibody to EL

Assay buffer containing 15 μl of anti-EL antibody was added toanti-mouse IgG-Fc antibody-immobilized microtiter plate, and incubatedfor 2 hr. After washing the wells three times with 90 μL of washingbuffer, 15 μl of cynomolgus monkey EL, baboon EL, rabbit EL, mouse EL,human [1-157]-mouse chimeric EL, human [1-305]-mouse chimeric EL orhuman [202-500]-mouse chimeric EL heparin extract was added andincubated at 4° C. for 16 hr. After washing the wells three times with90 μL of washing buffer, 15 μl of assay buffer containing biotin-labeledanti-C2-tag antibody and HRP-labeled Streptavidin were added to thewells and incubated at room temperature for 1 hr. After washing thewells three times with 90 μL of washing buffer, 15 μL ofTMB+—Substrate—Chromogen (DAKO) was added and incubated at roomtemperature for 30 min and then 15 μL of 0.05 M H2SO₄ was added andabsorbance at 450 nm was measured (FIG. 3A-3K).

As a result, 55A1, 7D4, 14A1, 2D5, 53A11, 13B3 and 23H8 bound cynomolgusmonkey EL, baboon EL and human [1-157]-mouse chimeric EL, but did notbind mouse EL. In addition, 13B3 and 23H8 bound rabbit EL, but 55A1,7D4, 14A1 2D5 and 53A11 did not. On the other hand, 16B3, 16E7, 14E1 and19E7 bound cynomolgus monkey EL, baboon EL, human [1-305]-mouse chimericEL and human [202-500]-mouse chimeric EL, but did not bind mouse El andhuman [1-157]-mouse chimeric EL. In addition, 16B3, 14E1 and 19E7 boundrabbit EL, but 16E7 did not.

Example 9

Binding Affinities of Anti-EL Antibodies

The binding affinities of anti-EL antibodies were measured usingBiacore. Anti-C2-tag antibody was immobilized on a sensor chip CM5 (GEHealthCare) using amine-coupling and baboon EL, cynomolgus monkey EL orhuman [1-305]-mouse chimeric EL heparin extract was added and EL wascaptured on the sensor chip. Then, anti-EL antibodies were added and thebinding affinities were calculated by bivalent fitting of BIAevaluationsoftware. The results were summarized in Table 1-3.

TABLE 1 Affinity for Baboon EL Clone name ka [1/Ms] kd [1/s] K_(D) [M]55A1 3.5 × 10⁵ 4.2 × 10⁻⁴ 1.2 × 10⁻⁹ 7D4 7.8 × 10⁴ 3.5 × 10⁻⁴ 4.5 × 10⁻⁹14A1 1.9 × 10⁵ 6.2 × 10⁻⁴ 3.3 × 10⁻⁹ 2D5 7.7 × 10⁵ 1.7 × 10⁻⁴ 2.2 × 10⁻⁹53A11 1.7 × 10⁵ 2.6 × 10⁻⁴ 1.6 × 10⁻⁹ 13B3 6.1 × 10⁴ 3.1 × 10⁻⁴ 5.1 ×10⁻⁹ 23H8 1.5 × 10⁵ 4.4 × 10⁻⁴ 2.9 × 10⁻⁹ 16B3 3.1 × 10⁴ 9.8 × 10⁻⁵ 3.2× 10⁻⁹ 16E7 5.3 × 10⁴ 2.5 × 10⁻⁴ 4.7 × 10⁻⁹ 14E1 1.5 × 10⁵ 8.5 × 10⁻⁴5.8 × 10⁻⁹ 19E7 6.7 × 10⁴ 8.3 × 10⁻⁴ 1.2 × 10⁻⁹

TABLE 2 Affinity for Cynomolgus EL Clone name ka [1/Ms] kd [1/s] K_(D)[M] 55A1 2.5 × 10⁵ 4.4 × 10⁻⁴ 1.7 × 10⁻⁹ 7D4 1.2 × 10⁵ 2.3 × 10⁻⁴ 1.9 ×10⁻⁹ 14A1 2.1 × 10⁵ 8.1 × 10⁻⁴ 3.9 × 10⁻⁹ 2D5 7.4 × 10⁵ 2.8 × 10⁻⁴ 3.8 ×10⁻⁹ 53A11 9.1 × 10⁵ 3.8 × 10⁻⁴ 4.2 × 10⁻⁹ 13B3 4.7 × 10⁴ 4.1 × 10⁻⁴ 8.7× 10⁻⁹ 23H8 1.2 × 10⁵ 7.5 × 10⁻⁴ 6.1 × 10⁻⁹ 16B3 4.2 × 10⁵ 8.9 × 10⁻⁵2.1 × 10⁻⁹ 16E7 3.2 × 10⁴ 3.2 × 10⁻⁴ 3.7 × 10⁻⁹ 14E1 1.2 × 10⁵ 6.9 ×10⁻⁴ 5.9 × 10⁻⁹ 19E7 5.5 × 10⁴ 1.8 × 10⁻⁴ 3.3 × 10⁻⁹

TABLE 3 Affinity for Human[1-305]-mouse chimeric EL Clone No. name ka[1/Ms] kd [1/s] K_(D) [nM] 1 55A1 1.9 × 10⁵ 1.4 × 10⁻⁴ 7.3 × 10⁻¹⁰ 2 7D41.1 × 10⁵ 1.1 × 10⁻⁴ 9.7 × 10⁻¹⁰ 3 14A1 2.1 × 10⁵ 1.0 × 10⁻⁴ 5.0 × 10⁻¹⁰4 2D5 8.5 × 10⁴ 9.3 × 10⁻⁵ 1.1 × 10⁻⁹  5 53A11 1.8 × 10⁵ 1.1 × 10⁻⁴ 6.3× 10⁻¹⁰ 6 13B3 1.5 × 10⁵ 1.4 × 10⁻⁴ 9.1 × 10⁻¹⁰ 7 23H8 1.2 × 10⁵ 1.1 ×10⁻⁴ 8.7 × 10⁻¹⁰ 8 16B3 5.1 × 10⁴ 1.9 × 10⁻⁴ 3.7 × 10⁻⁹  9 16E7 9.5 ×10⁵ 1.0 × 10⁻⁴ 1.1 × 10⁻⁹  10 14E1 4.2 × 10⁴ 2.3 × 10⁻⁴ 5.4 × 10⁻⁹ 

From the results of Example 5, 6, 8 and 9, the characteristics ofanti-EL antibodies were summarized in Table 4.

TABLE 4 Neutralizing activity IC50 [nM] a region of EL Clone baboonhuman-mouse cynomolgus rabbit which contributes No. name Subclass ELchimeric EL monkey EL EL to antibody binding 1 55A1 IgG2a 2.0 1.1 1.5N.T.  1-157 2 7D4 IgG1 2.6 3.7 2.1 N.T.  1-157 3 14A1 IgG1 2.7 5.8 3.1N.T.  1-157 4 2D5 IgG2a 2.9 2.7 3.0 N.T.  1-157 5 53A11 IgG2a 2.4 2.31.9 N.T.  1-157 6 13B3 IgG2a 2.6 4.1 1.7 13  1-157 7 23H8 IgG2a 3.2 1.72.7 67  1-157 8 16B3 IgG2a 3.0 1.3 1.9 6.3 202-305 9 16E7 IgG1 2.1 1.12.5 >40 202-305 10 14E1 IgG1 3.3 1.4 2.4 5.9 202-305 11 19E7 IgG2a 2.02.4 1.7 5.7 202-305

Example 10

Generation of Anti-EL Antibody which Neutralizes Both Human and Mouse EL

The adenovirus which expresses mouse EL was obtained using the samemethod as Example 1 and immunized to EL knock-out mice. In the samemanner as Example 4 and 5, anti-EL antibody (16F6) was achieved andsubclass was IgG1. The neutralizing activity and binding affinity of16F6 were measured and described in Table 5 and 6.

TABLE 5 Clone neutralizing activity name EL IC50 [nM] 16F6 baboon EL 2.8human (1-305) 4.0 mouse chimeric EL mouse EL 2.5 rabbit EL 2.3

TABLE 6 Clone Affinity name EL ka [1/Ms] kd [1/s] K_(D) [M] 16F6 human[1-305] 3.3 × 10⁵ 4.0 × 10⁻³ 1.4 × 10⁻⁸  mouse chimeric EL mouse EL 2.2× 10⁵ 1.5 × 10⁻⁴ 6.8 × 10⁻¹⁰ baboon EL 2.4 × 10⁵ 1.0 × 10⁻⁴ 4.2 × 10⁻¹⁰

Example 11

Determination of EL Regions which Contribute to Antibody Binding

The EL region which contributes to antibody binding was determined bythe homology-scanning mutagenesis or alanine scanning mutagenesis. Thebinding activity of anti-EL antibody to EL mutant was analyzed by ELISA.The amino acid residues of baboon EL were mutated to alanine usingQuikChange Site-Directed Mutagenesis Kit (Agilent Technologies), but theamino acid residues which were different from baboon EL and mouse ELwere mutated to mouse EL amino acid residues. Mutated EL heparinextracts were prepared and binding activity to anti-EL antibodyaccording to Example 2 and Example 8. The relative effect of eachmutation was evaluated from the following formula; ([antibodyconcentration where A450 value showed 1.0]-mutated EL)/([antibodyconcentration where A450 value showed 1.0]-wild type EL). The relativebinding activity of mutated baboon EL to anti-EL antibodies (55A1, 7D4,14A1, 2D5, 53A11, 13B3, 23H8, 16B3, 16E7, 14E1 and 19E7) was showed inFIG. 7A-L.

From the information of FIG. 7A-7G, it was shown that important aminoacids in the binding of the 55A1, 7D4, 14A1, 2D5, 53A11, 13B3 or 23H8antibody and EL are arginine at position 50, glutamic acid at position60, histidine at position 61, tyrosine at position 65 and asparagine atposition 100 in an amino acid sequence of SEQ ID NO: 1.

From the information of FIG. 7H-7K, it was shown that important aminoacids in the binding of the 16B3, 16E7, 14E1 or 19E7 antibody and EL arehistidine at position 220, threonine at position 221, tyrosine atposition 222, threonine at position 223, arginine at position 224,phenylalanine at position 226, glycine at position 227, glycine atposition 231, isoleucine at position 232, glutamine at position 233,methionine at position 234, aspartic acid at position 240, tyrosine atposition 242, proline at position 243, asparagine at position 244,glycine at position 246, glutamine at position 249, proline at position250, glycine at position 251, leucine at position 254, leucine atposition 258, tyrosine at position 263, valine at position 269 andglutamic acid at position 273 in an amino acid sequence of SEQ ID NO: 1.

Example 12

Analysis of Amino Acid Sequence of Variable Region of Anti-EL Antibodies

The amino acid sequence of variable region of anti-EL antibody wasdetermined using conventional procedure and described in FIG. 4A-L.

Example 13

Alignment of Amino Acid Sequences of Variable Region of 55A1, 7D4, 2D5,53A11, 13B3 and 23H8

The amino acid sequences of variable region of 55A1, 7D4, 2D5, 53A11,13B3 and 23H8 were aligned by GENETYX software (FIG. 5A˜D). FIGS. 5A and5B indicated that all six CDRs of heavy and light chain of 55A1, 7D4,14A1 and 2D5 had high similarity. FIG. 5C indicated that CDR1 and CDR2of heavy chain of 53A11, 13B3 and 23H8 had high similarity.

The analysis of CDRs of heavy and light chain of 55A1, 7D4, 14A1 and 2D5revealed the following things.

The amino acid sequences of heavy chain CDR1 was consisted of 5 aminoacids, NYGMN (SEQ ID NO: 10).

The amino acid sequences of heavy chain CDR2 was consisted of 17 aminoacids, WINTYSGVPTY (A or T) (G or D) DFKG (SEQ ID NO: 107).

The amino acid sequences of heavy chain CDR3 was consisted of 11 aminoacids, (R or F) (G or S) YYGR (R or H) YFD (V or Y) (SEQ ID NO: 108).

The amino acid sequences of light chain CDR1 was consisted of 15 aminoacids, KASQSVDYD (V or G) DSYM (H or N) (SEQ ID NO: 109).

The amino acid sequences of light chain CDR2 was consisted of 7 aminoacids, AASNLXS (SEQ ID NO: 110). “X” represents given amino acid.

The amino acid sequences of light chain CDR3 was consisted of 10 aminoacids, (H or Q) Q (T or S) (I or T or N) (E or D) DPP (- or W) T (SEQ IDNO: 111). “-” represents deletion of amino acid.

The analysis of CDRs of heavy and light chain of 53A11, 13B3 and 23H8revealed the following things.

The amino acid sequences of heavy chain CDR1 was consisted of 5 aminoacids, (D or E) (Y or N) T (M or I) H (SEQ ID NO: 112).

The amino acid sequences of heavy chain CDR2 was consisted of 17 aminoacids, XINPYYGGTX (Y or N) NEKFKD (SEQ ID NO: 113). “X” represents givenamino acid.

There was no commonality of amino acid residues in heavy chain CDR3.

Example 14

Alignment of Amino Acid Sequences of Variable Region of 16B3, 16E7, 14E1and 19E7

The amino acid sequences of variable region of 16B3, 16E7, 14E1 and 19E7were aligned by GENETYX software (FIG. 6A˜D). FIGS. 6A and 6B indicatedthat all six CDRs of heavy and light chain of 16B3 and 16E7 had highsimilarity.

The analysis of CDRs of heavy and light chain of 16B3, 16E7, 14E1 and19E7 revealed the following things.

The amino acid sequences of heavy chain CDR1 was consisted of 5 aminoacids, (G or S) YTMS (SEQ ID NO: 114).

The amino acid sequences of heavy chain CDR2 was consisted of 17 aminoacids, EISF (A or T) R (D or S) RAFYPDTVKG (SEQ ID NO: 115).

The amino acid sequences of heavy chain CDR3 was consisted of 13 aminoacids, LGG (R or N) N (H or Y) DYWYFDV (SEQ ID NO: 116).

The amino acid sequences of light chain CDR1 was consisted of 15 aminoacids, RASESVEYYGTSLMQ (SEQ ID NO: 70 or 78).

The amino acid sequences of light chain CDR2 was consisted of 7 aminoacids, AASNVES (SEQ ID NO: 71 or 79).

The amino acid sequences of light chain CDR3 was consisted of 9 aminoacids, QQSWKVPFT (SEQ ID NO: 72 or 80).

INDUSTRIAL APPLICABILITY

The monoclonal antibody which inhibits the enzymatic activity ofvascular endothelial lipase of the present invention is useful as a drugfor prevention and/or treatment of dyslipidemia, hyperlipidemia,arteriosclerosis, atherosclerosis, hypercholesterolemia,hypertriglyceridemia, diabetes, obesity and/or syndrome X because it hasselectively inhibitory activity against vascular endothelial lipase.

Sequence List

1. A method of inhibiting endothelial lipase (EL) activity, said methodcomprising: contacting EL with a monoclonal antibody or an antibodyfragment thereof, wherein said antibody or fragment thereof recognizesat least four amino acids at positions of 50 to 100 in the amino acidsequence of SEQ ID NO: 1 including: 1) tyrosine at position 65, and 2)at least three amino acids selected from arginine at position 50,asparagine at position 52, arginine at position 54, aspartic acid atposition 58, glutamic acid at position 60, histidine at position 61,glycine at position 63, and asparagine at position
 100. 2. The methodaccording to claim 1, wherein said antibody fragment is a Fab, F(ab′)2,Fab′, scFv, dsFv or Diabody.
 3. The method according to claim 1, whereinsaid monoclonal antibody or fragment thereof has a heavy chain variableregion comprising amino acid sequence SEQ ID NO: 9, and a light chainvariable region comprising amino acid sequence SEQ ID NO:
 13. 4. Themethod according to claim 1, wherein said antibody or fragment thereofrecognizes at least tyrosine at position 65, asparagine at position 52,arginine at position 54, glutamic acid at position 60, glycine atposition 63, and asparagine at position 100 in the amino acid sequenceof SEQ ID NO:
 1. 5. The method according to claim 1, wherein saidantibody or fragment thereof recognizes at least tyrosine at position65, asparagine at position 52, glycine at position 63, and asparagine atposition 100 in the amino acid sequence of SEQ ID NO:
 1. 6. The methodaccording to claim 1, wherein said antibody or fragment thereofrecognizes at least tyrosine at position 65, arginine at position 54,glycine at position 63, and asparagine at position 100 in the amino acidsequence of SEQ ID NO:
 1. 7. The method according to claim 1, whereinsaid antibody or fragment thereof recognizes at least tyrosine atposition 65, glutamic acid at position 60, glycine at position 63, andasparagine at position 100 in the amino acid sequence of SEQ ID NO: 1.8. A method of binding to endothelial lipase (EL) with a monoclonalantibody or an antibody fragment thereof, said method comprising:contacting EL with a monoclonal antibody or an antibody fragmentthereof, wherein said antibody or fragment thereof recognizes at leastfour amino acids at positions of 50 to 100 in the amino acid sequence ofSEQ ID NO: 1 including: 1) tyrosine at position 65, and 2) at leastthree amino acids selected from arginine at position 50, asparagine atposition 52, arginine at position 54, aspartic acid at position 58,glutamic acid at position 60, histidine at position 61, glycine atposition 63, and asparagine at position
 100. 9. The method according toclaim 8, wherein said antibody fragment is a Fab, F(ab′)2, Fab′, scFv,dsFv or Diabody.
 10. The method according to claim 8, wherein saidmonoclonal antibody or fragment thereof has a heavy chain variableregion comprising amino acid sequence SEQ ID NO: 9, and a light chainvariable region comprising amino acid sequence SEQ ID NO:
 13. 11. Themethod according to claim 8, wherein said antibody or fragment thereofrecognizes at least tyrosine at position 65, asparagine at position 52,arginine at position 54, glutamic acid at position 60, glycine atposition 63, and asparagine at position 100 in the amino acid sequenceof SEQ ID NO:
 1. 12. The method according to claim 8, wherein saidantibody or fragment thereof recognizes at least tyrosine at position65, asparagine at position 52, glycine at position 63, and asparagine atposition 100 in the amino acid sequence of SEQ ID NO:
 1. 13. The methodaccording to claim 8, wherein said antibody or fragment thereofrecognizes at least tyrosine at position 65, arginine at position 54,glycine at position 63, and asparagine at position 100 in the amino acidsequence of SEQ ID NO:
 1. 14. The method according to claim 8, whereinsaid antibody or fragment thereof recognizes at least tyrosine atposition 65, glutamic acid at position 60, glycine at position 63, andasparagine at position 100 in the amino acid sequence of SEQ ID NO: 1.